A more comprehensive understanding of the mechanisms that control normal tissue-and developmental stage-specific expression of human beta globin gene family is essential for the development of new treatment strategies. We have now generated a transgenic model of human beta globin gene regulation during ontogeny by transfer of the intact human beta globin locus in Yeast Artificial Chromosomes (YACs). Our long-term goal is to use the beta globin YAC transgenic model to more precisely define the role of cis-acting regulatory sequences that direct tissue- and stage-specific expression of the human beta globin gene family. Our hypothesis is that multiple sequence elements involved in the developmental program of beta gene expression reside within, or immediately flanking the individual genes and their promoters. To test this hypothesis, we will make mutations in putative regulatory regions using homology-directed recombination, and analyze their effect on the regulation of human gamma and beta globin gene expression. We will also develop a complementary strategy by making mutations in the human beta globin locus in embryonic stem (ES) cells lines derived from our transgenic lines carrying a single copy of the human beta globin locus. The ES cell-based approach will allow us to directly compare the effects of each of the mutations in a transgenic human beta globin locus with the same chromosomal position of integration. Transgenic mice and ES cell lines carrying the human beta globin locus will also be valuable for assessing the role of known trans-acting factors and will provide unique models for defining the function of newly identified genetic loci in regulating the human beta globin gene family. Our analysis will provide a more complete understanding of the sequence elements required for globin gene regulation during development, and may ultimately assist in the development of novel genetic and pharmacological approaches to the treatment of SCD.